Crystalline N-acetyl neuraminic acid derivatives and processes for their preparation

ABSTRACT

Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonic acid in crystaline form, particularly in the form of a hydrate which exists as crystals having a low aspect ratio, or in the form of a hydrate which exists as crystals having a high aspect ratio.

[0001] The present invention relates to derivatives of N-acetylneuraminic acid and their use in medicine. More particularly theinvention is concerned with particular physical forms of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (the 4-guanidino analogue of DANA; also known as5-(acetylamino)-2,6anhydro-3,4,5-trideoxy-4-guanidino-D-glycereo-2-galacto-non-2-enonicacid), pharmaceutical formulations thereof and their use in therapy.

[0002] PCT/AU91/00161 (publication no. WO91/16320) describes a number ofderivatives of5-acetamidino-2,3,5-trideoxy-4-D-glycero-D-galacto-non-2-enopyranosonicacid (2,3,-dideoxy-2,3-didehydro-N-acetyl-neuraminic acid; DANA)including the 4-guanidino analogue of DANA. The 4-guanidino analogue ofDANA is prepared by the reaction of the corresponding O-acyl protected4-amino analogue of DANA by reaction with S-methylisourea followed bydeprotection, purification by chromatography and freeze-drying.

[0003] The structure of the 4-guanidino analogue of DANA is shown below:

[0004] We have now found that the compound of formula (I) can beobtained in crystalline form.

[0005] There is thus provided in a first aspect of the invention5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form.

[0006] We have further found that the compound of formula (I) may beobtained by crystallisation under certain conditions in the form of acrystalline hydrate (hereinafter Hydrate I). Hydrate I exists in theform of crystals having a low aspect ratio, for example, tabularcrystals, which are favoured for pharmaceutical formulation because oftheir physical properties, e.g. good flow characteristics. The watercontent of Hydrate I is related to relative humidity (RH). Water uptakeof Hydrate I varies from zero at RH of 0% up to 10% at RH of 90-100%.

[0007] The compound of formula (I) may also be crystallised in the formof a dihydrate (hereinafter Hydrate II). Hydrate II exists in the formof crystals having a high aspect ratio, for example, needle-shapedcrystals. The water content of these crystals remains substantiallyconstant over a broad relative humidity range (RH about 10-90%). Thestable water content of Hydrate II represents an advantage of thiscrystalline form for use in pharmacy.

[0008] There is thus provided in a further aspect of the invention5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-2-galacto-non-2-enopyranosonicacid in the form of crystals having a low aspect ratio, such as tabularcrystals.

[0009] In a further aspect there is provided5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of crystals having a high aspect ratio, such asneedle-shaped crystals.

[0010] Whilst tabular crystals are regarded as typical of Hydrate I andneedle-shaped crystals are regarded as typical of Hydrate II, it will beappreciated that the possibility of either Hydrate I or Hydrate IIexisting in alternative crystal habits under certain circumstancescannot be excluded. It is to be understood that all such alternativecrystal habits are within the scope of the present invention.

[0011] There is also provided5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of crystals which have stable water content over abroad humidity range, for example RH 10-90%.

[0012] Hydrate I loses substantially all of its water of crystallisationat about 80-90° C. Decomposition occurs at 299° C.

[0013] Hydrate II loses one mole of water of crystallisation at about84-90° C. and a further mole of water of crystallisation by about135-143° C.

[0014] In a yet further aspect the invention provides5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of a crystalline hydrate which loses substantially allits water of crystallisation at 80-90° C.

[0015] In a yet further aspect the invention provides5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of a crystalline hydrate which loses one mole of waterof crystallisation at 84-90° C. and a further mole of water ofcrystallisation at 135-143° C.

[0016] In a preferred aspect the invention provides5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of Hydrate I as herein defined substantially free ofHydrate II as herein defined.

[0017] In a further preferred aspect the invention provides5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of Hydrate II as herein defined substantially free ofHydrate I as herein defined.

[0018] By “substantially free” is meant containing less than 5% of thealternative hydrate, such as less than 2%, for example less than 1% ofthe alternative hydrate.

[0019]5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid may be prepared in crystalline form by crystallisation of thecompound from aqueous solution.

[0020] Each of Hydrate I and Hydrate II may be prepared substantiallyfree from the alternative Hydrate by controlling the solutionconcentration and temperature at which crystallisation occurs.

[0021] In general,5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of Hydrate I may be obtained by crystallisation of thecompound from aqueous solution at a temperature greater than about 50°C., preferably 50-55° C.

[0022] In general,5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of Hydrate II may be obtained by crystallisation of thecompound from aqueous solution at a temperature below about 40° C.,preferably about 20-30° C.

[0023] Crystallisation of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid from aqueous solution at a temperature in the range of about 40-50°C. typically results in a mixture of tabular and needle-shaped crystals.Such mixtures are disfavoured for the preparation of pharmaceuticalformulations because of the differing physical properties of Hydrate Iand Hydrate II, in particular their flow properties.

[0024] Seeding of an aqueous solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid with crystals of Hydrate I or Hydrate II may lead tocrystallisation of the seeded Hydrate. Preparation of Hydrate I orHydrate II should therefore be conducted in the absence of seeds of theundesired Hydrate. Conversely, Hydrate I may be prepared by seeding anaqueous solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid with crystals of Hydrate I, and Hydrate II may be prepared byseeding an aqueous solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid with crystals of Hydrate II.

[0025] For the preparation of Hydrate II it is preferable to employ arelatively dilute aqueous solution, for example a solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in 15-30 volumes of water, for example 20 volumes of water. HydrateI may conveniently be crystallised from a relatively concentratedaqueous solution, for example a solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonic acid in 12-20volumes of water, such as 12-15 volumes of water.

[0026] We have found that Hydrate II may be converted into Hydrate I inaqueous suspension or saturated solution. Such interconversion may beeffected by prolonged ageing of an aqueous suspension or saturatedsolution of Hydrate II, for example ageing for a period of days, forexample more than 10 days, such as about 15 days. Alternatively,interconversion may be effected in the presence of a base, for examplean organic base such as imidazole.

[0027] Recovery of either Hydrate I or Hydrate II from aqueous solutionmay be enhanced by the addition to the solution of a suitablecounter-solvent. Suitable counter-solvents are water-miscible solventsin which the compound of formula (I) has poor solubility. Convenientlythe counter-solvent will be a ketone, such as acetone, or an alkanolsuch as propan-2-ol. A preferred counter-solvent is acetone.

[0028] We have also found that addition of an aqueous solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid to a similar volume of a counter-solvent as previously definedresults in precipitation of Hydrate II. For example, addition of asolution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in 12-15 volumes of water to 12-20 volumes of acetone givescrystals of Hydrate II.

[0029] The methods for the preparation of crystalline material, and inparticular methods for the preparation of Hydrate I and Hydrate II,described herein constitute further aspects of the present invention.

[0030]5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form may be used as an antiviral agent as describedin WO 91116320, which is incorporated herein by reference.

[0031]5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form may be formulated as a pharmaceuticalcomposition for use as an antiviral agent as described in WO 91/16320.

[0032] Preferred pharmaceutical formulations of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid include powder formulations and aqueous solutions or suspensions.Preparation of powder formulations requires micronisation of the drugsubstance. The good flow properties of Hydrate I render it particularlysuitable for micronisation. Hydrate II has adequate flow properties andalso a particularly rapid dissolution rate in water. These propertiesrender Hydrate II particular advantageous for the preparation of aqueoussolutions/suspensions.

[0033] Hydrates I and II have been subjected to X-ray powder diffractionstudies. Diffraction traces were obtained using a Seimens D-500diffractometer and CuK₆₀ radiation. X-Ray intensities were measured at0.020 increments for 5 second intervals using a scintillation counter,between values of 5 and 55° 2θ. The d-spacings and line intensitiesobtained for Hydrate I and Hydrate II are shown in Tables I and II,respectively. TABLE I d(A) I(%) 10.06 30.25 6.77 69.81 6.63 89.61 6.3512.69 6.05 54.56 5.38 25.11 5.05 98.58 4.61 12.58 4.42 100.00 4.31 8.284.17 11.67 3.98 75.00 3.90 52.61 3.77 20.33 3.69 36.17 3.48 26.53 3.4153.25 3.37 17.61 3.16 18.39 3.02 31.08 2.98 9.25 2.92 6.28 2.87 13.582.82 10.78 2.78 6.78 2.74 18.03 2.69 15.33 2.65 6.25 2.63 6.44 2.5911.44 2.49 14.31 2.45 18.81 2.41 8.64 2.35 11.36 2.19 5.42 2.13 12.252.11 6.56 2.02 8.33 1.98 5.47

[0034] TABLE II d(A) I(%) 16.88 66.34 10.38 50.60 9.50 16.08 8.47 40.467.12 100.00 5.84 11.78 5.33 18.83 5.21 33.99 4.78 12.94 4.57 75.81 4.3216.37 4.25 18.49 4.14 43.26 3.96 10.33 3.76 22.11 3.64 25.16 3.57 37.043.52 15.69 3.40 16.85 3.34 21.20 3.17 13.52 3.13 17.04 3.06 7.48 2.9410.19 2.92 8.45 2.86 9.17 2.76 9.56 2.72 9.22 2.67 6.81 2.64 8.06 2.605.46 2.58 6.52 2.51 5.31 2.49 6.66 2.45 5.55 2.43 5.89 2.39 15.93 2.3810.38 2.31 8.40 2.22 5.94 2.16 5.36 2.11 6.28 2.03 7.24 1.91 6.57

[0035] The following examples illustrate the invention but are notintended as a limitation thereof. All temperatures are in °C.

EXAMPLE 1

[0036] Preparation of Hydrate I

[0037] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (5.0 g) and water (60 ml) was heated at 100° to give a clearsolution. The solution was cooled during 30 min to 55° and maintained atbetween 55° and 50° during 4h, to give a crystalline suspension. Acetone(80 ml) was added during 90 min, the temperature being maintainedbetween 48 and 55°. The resultant slurry was stirred 1 h, thetemperature being allowed to fall to ca. 20°, and the suspension wasallowed to stand 17h at ambient temperature. The product was collectedby vacuum filtration and the filter bed was washed with 4:1acetone/water (2×10 ml) then with acetone (10 ml). The product was airdried at ambient temperature and humidity to give Hydrate I (tabularcrystals) (4.5 g).

[0038] PMR (D₂O) 2.04 (3H,s), 3.67 (2H,m), 4.23 (1H,m), 4.42 (2H,m),5.63 (1H, d, J, 2.5 Hz)

[0039] IR (Nujol) 3248, 3338, 3253; NH, OH 1692, 1666, 1646, 1619, 1575;CO (CH₃CONH, CO₂), CN

[0040] Water content 8.4% w/w; calculated for C₁₂H₂₀N₄O₇.1.7H₂O

EXAMPLE2

[0041] Preparation of Hydrate I

[0042] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (15.0 g) and water (180 ml) was heated at 100° to give a clearsolution. The solution was clarified by vacuum filtration through afilter paper then cooled to ca. 55° and maintained at between 55° and50° during 4h, allowing crystallisation to become established. Acetone(210 ml) was added with stirring, during 2h, the temperature beingmaintained at 48-55°. The resultant suspension was stirred and cooled to30° and was then allowed to stand at ambient temperature 17h. The solidwas filtered and the product was washed with 4:1 acetone/water (2×30 ml)and then acetone (30 ml). The solid was air dried at ambient temperatureand humidity to give Hydrate I (tabular crystals) (12.0 g).

[0043] Characterisation as above.

EXAMPLE 3

[0044] Preparation of Hydrate II

[0045] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (10.0 g) and water (100 ml) was heated to 95°. The resultantsolution was clarified by vacuum filtration. The solution was thencooled to 300 and acetone (250 ml) was added during 5 min stirring. Theresultant thick white suspension was allowed to stand at ambienttemperature 20 h. The solid was collected by vacuum filtration and waswashed with 4:1 acetone/water (2×20 ml) then with acetone (20 ml). Thesolid was dried in a vacuum oven at 35° for 24h and then equilibratedwith atmospheric moisture at ambient temperature and humidity to giveHydrate II (needle-shaped crystals) (8.16 g).

[0046] Characterisation as above.

[0047] Water content 10.6% w/w; calculated for C₁₂H₂₀N₄O₇.2H₂O 9.8% w/w

EXAMPLE 4

[0048] Preparation of Hydrate II

[0049] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (10.0 g) and water (400 ml) was heated to 20° for 2h. The resultantsolution was clarified by vacuum filtration. Acetone (110 ml) was addedand solid began to crystallise. The resultant suspension was stirred for2.5h at 20°. The solid was collected by vacuum filtration and was washedwith 4:1 v/v acetone/water (2×20 ml) then with acetone (20 ml). Thesolid was dried in a vacuum oven at 30° and then equilibrated withatmospheric moisture at ambient temperature and humidity to give HydrateII (need shaped crystals) (7.7 g)

[0050] Characterisation as above.

[0051] Water content 11.1% w/w calculated for C₁₂H₂₀N₄O₇.2H₂O 9.8 w/w.

EXAMPLE 5

[0052] Preparation of Hydrate II

[0053] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (50 g) and water (1150 ml) was heated to 75°. The resultantsolution was clarified by vacuum filtration, washing through with water(100 ml). The solution was then cooled to 7° over 1h and acetone (500ml) was added. The resultant solution was stirred slowly for 0.5h,during which time solid began to crystallise. Acetone (750 ml) was thenadded to the suspension over 2h, maintaining the temperature at 5-10°.The solid was collected by vacuum filtration and washed with 4:1 v/vacetone/water (2×100 ml) then with acetone (100 ml). The solid wasair-dried at ambient temperature and humidity to give Hydrate II(needle-shaped crystals) (46.0 g).

[0054] Water content 9.8% w/w; calculated for C₁₂H₂₀N₄O₇.2H₂O 9.8% w/w.

[0055] XRD: consistent with Hydrate II (>99%).

EXAMPLE 6

[0056] Precipitation of Hydrate II

[0057] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonicacid (50 g) and water (600 ml) was heated to 100°. The resultant clearhot solution was added over 8 minutes to acetone (700 ml) stirredrapidly at ambient temperature, causing the temperature of the mixtureto rise from 20° to 56° and precipitation of solid. The resultantsuspension was allowed to cool to 20° with stirring, then the solid wascollected by vacuum filtration and washed with 4:1 acetonetwater (2×100ml) then with acetone (100 ml). The solid was air-dried at ambienttemperature and humidity to give Hydrate II (needle-shaped crystals)(47.9 g).

[0058] Water content 10.0 w/w; calculated for C₁₂H₂₀N₄O₇.2H₂O 9.8% w/w.

[0059] XRD: consistent with Hydrate II (>99%).

EXAMPLE 7

[0060] Crystallisation of Hydrate I from Water

[0061] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonicacid (35.7 g) and water (350 ml) was heated at 95° to give a clearsolution. This was adjusted to pH7.0 (from pH6.4) with aqueous aceticacid (100 μp, 10%v/v). The resulting solution was allowed to cool toambient temperature with stirring, to give a crystalline suspension. Theproduct was collected by vacuum filtration, then vacuum-dried at ambienttemperature to give Hydrate I (tabular crystals) (28.9 g).

[0062] IR consistent with Hydrate I

EXAMPLE 8

[0063] Preparation of Hydrate II by Seeding

[0064] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonicacid (30.0 g) and water (540 ml) was heated at 75° to give a solution.The resultant solution was clarified by vacuum filtration, washingthrough with water (54 ml). The solution was heated to 100°, cooled to40°, then seeded with Hydrate II (0.3 g). Crystallisation occurred asthe temperature was further reduced to ambient temperature. Theresultant slurry was stirred for 1h at ambient temperature, cooled to5°, then acetone (600 ml) was added over 1.5h. The solid was collectedby vacuum filtration and washed with 4:1 v/v acetone/water (2×60 ml)then with acetone (60 ml). The solid was air-dried at ambienttemperature and humidity to give Hydrate II (needle-shaped crystals)(26.5 g).

[0065] XRD 90-95% Hydrate II (5-10% Hydrate I)

EXAMPLE 9

[0066] Interconversion of Hydrate II to Hydrate I by Ageing

[0067] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonicacid (10.0 g) and water (200 ml) was heated at 100° to give a solution.The resultant solution was rapidly cooled to 30°, seeded with Hydrate II(0.059) then left unstirred overnight. Light-microscopy showedexclusively the characteristic needles of Hydrate II. The suspension wasaged unstirred at ambient temperature for 11 days (when light-microscopyshowed the presence of some crystals of Hydrate I), then stirred for 3days. Acetone (200 ml) was added, and the slurry was stirred for 1h. Thesolid was collected by vacuum filtration and washed with 4:1 v/vacetone/water (2×20 ml) then with acetone (20 ml). The solid wasair-dried at ambient temperature and humidity to give Hydrate I (tabularcrystals) (9.0 g).

[0068] XRD: Consistent with Hydrate I>99%)

EXAMPLE 10

[0069] Interconversion of Hydrate II to Hydrate I using Base

[0070] A suspension of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid (5.0 g, Hydrate II) in water (30 ml), containing imidazole (2.96 g)was stirred and heated at 30° for 40h. The remaining solid was collectedby vacuum filtration and washed with water (2×1 ml, 2×5 ml) thenair-dried at ambient temperature and humidity to give Hydrate I (tabularcrystals) (3.98 g).

[0071] IR consistent with Hydrate I

EXAMPLE 11

[0072] Preparation of Hydrate I by Seeding

[0073] A mixture of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonicacid (5.0 g) and water (60 ml) was heated at 100° to give a solution.The resultant solution was clarified by vacuum filtration. The resultantsolution was cooled to about 50°, seeded with Hydrate I, left unstirredat 50-55° for 1h, then stirred for 1h at 50-55°. Acetone (70 ml) wasadded whilst a temperature of 48-55° was maintained. The slurry wasstirred for 1h at 50-55°, then aged unstirred overnight at ambienttemperature. The solid was collected by vacuum filtration and washedwith 4:1 v/v acetone/water (2×10 ml) then with acetone (2×10 ml). Thesolid was vacuum-dried, then allowed to re-equilibrate at ambienttemperature and humidity to give Hydrate I (tabular crystals) (3.8 g).

[0074] XRD: Consistant with Hydrate I (>99%)

1.5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form. 2.5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of crystals having a low aspect ratio.
 3. Thecrystalline form as claimed in claim 2 wherein the crystals are tabular.4. The crystalline form as claimed in any one of claims 1 to 3 whereinsubstantially all water of crystallisation is lost at about 80 to 90° C.5. The crystalline form as claimed in any one of claims 1 to 4 for whichthe X-ray data are as shown in Table I. 6.5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in the form of crystals having a high aspect ratio.
 7. Thecrystalline form as claimed in claim 6 wherein the crystals areneedle-shaped.
 8. The crystalline form as claimed in any one of claims1, 6 or 7 wherein water content is stable over a broad range of relativehumidity.
 9. The crystalline form as claimed in any one of claims 1, 6to 8 wherein one mole of water of crystallisation is lost at about84-90° C. and a further mole of water of crystallisation is lost byabout 135-143° C.
 10. The crystalline form as claimed in any one ofclaims 1, 6 to 9 for which the X-ray data are as shown in Table II. 11.The crystalline form claimed in any one of claims 2 to 5 substantiallyfree of the crystalline form claimed in any one of claims 6 to
 10. 12.The crystalline form claimed in any one of claims 6 to 10 substantiallyfree of the crystalline form claimed in any one of claims 2 to
 5. 13. Amethod for the preparation of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form, which method comprises crystallisation of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid from aqueous solution.
 14. A method as claimed in claim 13 for thepreparation of the crystalline form as claimed in any one of claims 2 to5.
 15. A method as claimed in claim 14 wherein the temperature of theaqueous solution is greater than about 50° C.
 16. A method as claimed inclaim 15 wherein the temperature of the aqueous solution is in the range50 to 55° C.
 17. A method as claimed in any one of claims 14 to 16wherein the aqueous solution is seeded with crystals of the crystallineform as claimed in any one of claims 2 to
 5. 18. A method as claimed inclaim 13 for the preparation of the crystalline form as claimed in anyone of claims 6 to
 10. 19. A method as claimed in claim 18 wherein thetemperature of the aqueous solution is less than about 40° C.
 20. Amethod as claimed in claim 19 wherein the temperature of the aqueoussolution is in the range 20 to 30° C.
 21. A method as claimed in any oneof claims 18 to 20 wherein the aqueous solution is seeded with crystalsof the crystalline form as claimed in any one of claims 6 to
 10. 22. Amethod as claimed in any one of claims 13 to 21 comprising addition of acounter solvent to the aqueous solution.
 23. A method as claimed inclaim 22 wherein the counter solvent is a ketone or an alkanol.
 24. Amethod as claimed in claim 23 wherein the counter solvent is acetone.25. A method for the preparation of the crystalline form as claimed inany one of claims 2 to 5, which method comprises interconversion of thecrystalline form as claimed in any one of claims 6 to
 10. 26. The methodas claimed in claim 25 wherein interconversion is effected by ageing ofthe aqueous solution.
 27. The method as claimed in claim 25 wherein theinterconversion is effected by addition of a base to the aqueoussolution.
 28. A method for the preparation of the crystalline form asclaimed in any one of claims 6 to 10, which process comprises additionof an aqueous solution of5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid to a similar volume of a counter solvent.
 29. The method as claimedin claim 28 wherein the counter solvent is acetone.
 30. A pharmaceuticalformulation comprising5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-enopyranosonicacid in crystalline form and a pharmaceutically acceptable carriertherefor.
 31. A pharmaceutical formulation as claimed in claim 30 in theform of a powder.
 32. A pharmaceutical formulation as claimed in claim30 in the form of an aqueous solution or suspension.